The aim of this study was to clone and construct a buffalo miR-302s (bbu-miR-302s) lentivirus expression vector,analyze its bioinformatics,and try to apply the vector to buffalo somatic cell reprogramming.The bbu-miR-302s precursor sequence was amplified using buffalo genomic DNA as template,after the TA clone was sequenced,it was ligated into pLVX-IRES-ZsGreen1 lentiviral expression vector.After the vector was identified by enzyme digestion,it was transferred into HEK-293T cells by lipofection to package the lentivirus,and bbu-miR-302s lontivirus infect the HEK-293T and fibroblast cells of pig and buffalo.After buffalo fetal fibroblasts (BFFs) were infected by bbu-miR-302s lentivirus,the production of buffalo induced pluripotent stem cells (iPSC) was detected by induction culture.The location analysis of the miR-302s family in the genome was performed using the CoGeMiR database and SnapGene Viewer,its conservative was analyzed using ClustalX 1.83 software;TargetScan and miRWalk were used to predict the major target genes,and DAVID program was used to enrich the signal pathway of target genes.The sequencing results showed that the amplified sequence was buffalo miR-302s family,the titer of the packaged lentivirus was 7.2×10⁶ TU/mL,which could effectively infect the somatic cells of three species.After BFFs were infected by bbu-miR-302s lentivirus,the cells changed morphologically and gathered to form clones.Alkaline phosphatase was positive,but the results of pluripotency gene and surface antigen were negative,which indicated that single factor miR-302s was not enough to completely reprogram BFF into iPSC,it promoted the reprogramming process.The CoGeMiR database showed that the miR-302s family was mainly located in the intron region of LARP7 gene,SnapGene Viewer software analysis result showed that bbu-miR-302s was located in the intron region of LARP7 gene in buffalo chromosome 7.The miR-302s clusters of different species and members of miR-302s family were highly conserved.A total of 255 major target genes of buffalo miR-302s were predicted,and these target genes were mainly concentrated in 33 pathways,among which PGK signal pathway,glucagon signal transduction and regulation of stem cell signal pathway were the most significant.The results laid a foundation for further research on the role of the miR-302s cluster in somatic cell reprogramming.

To study the role of dnaJ gene in the pathogenicity of piscine S.agalactiae,isogenic mutant (ΔdnaJ) and complementation strain (CΔdnaJ) of dnaJ gene were respectively constructed.The primers were designed according to the target gene sequence in GenBank of S.agalactiae GD201008-001(accession No.:NC_018646).The upstream and downstream homologous arm sequences of dnaJ gene were amplified and fused by PCR.dnaJ gene containing promoter sequence was also amplified by PCR.The upstream and downstream homologous arm fusion fragment of dnaJ gene and dnaJ gene containing promoter sequence were cloned into the Streptococcus-Escherichia coli shuttle plasmids pSET4s and pSET2,respectively.The recombinant plasmid was electrotransformed into GD201008-001 competent cells to construct ΔdnaJ and CΔdnaJ.By analyzing genetic stability and morphological changes of ΔdnaJ,CΔdnaJ and wild-type strain,effects of deletion of dnaJ gene on the transcription of downstream and downstream genes,growth rate and bacterial virulence in a zebrafish infection model,the effects of dnaJ gene on the virulence of Streptococcus agalactis were evaluated.PCR identification and sequencing showed that ΔdnaJ and CΔdnaJ were successfully constructed.ΔdnaJ and CΔdnaJ showed no significant differences in bacterial morphology compared with the wild-type strain,but the growth speed of ΔdnaJ decreased significantly.The deletion of dnaJ gene had no effect on the transcription of upstream and downstream genes.Furthermore,the 50% lethal dose of ΔdnaJ (5.68×10⁴ CFU) was increased up to 241-fold of the parental strain in a zebrafish infection model.These findings demonstrated that the dnaJ gene of piscine S.agalactiae exerted a significant effect on bacterial virulence.This results provided a reference basis for further exploration of dnaJ gene of S.agalactiae.

The sodium-dependent neutral amino acid transporter 2 (SNAT2) is an amino acid transporter which can transport neutral amino acids and is widely distributed in various cells.Amino acids is not only substrates for protein synthesis,but also key signal molecules regulating cell metabolism.However,it is little known whether SNAT2 mediates the regulation of amino acids on autophagy.In this study,CASY cell count and Western blotting were used to determine the proliferation of BMECs after SNAT2 overexpression and siRNA interference.The effects of SNAT2 on the expression of autophagic biomarker LC3-Ⅱ was detected by Western blotting and the change of autophagic spots (LC3-Ⅱ) in cells was determined by immunofluorescence.The results showed that SNAT2 overexpression increased the expression levels of p-PI3K,p-mTOR and cyclin D1,whereas SNAT2 knockdown had the opposite effects.Furthermore,the expression of LC3-Ⅱ and the autophagy spots were both increased in SNAT2-downregulated cells.The p-mTOR expression was increased and LC3-Ⅱ was decreased in cells treated with the autophagy enhancer trehalose dihydrate (Tre) together with methionine (Met) compared with the control (cells treated with Tre).The expression of p-mTOR was decreased and LC3-Ⅱ was increased in cells treated with Tre together with Met addition and transfected with a SNAT2 siRNA compared with the control (cells treated with Tre and Met).In conclusion,these data demonstrated that SNAT2 mediated the regulation of Met on proliferation of and autophagy in BMECs via PI3K-mTOR/Cyclin D1 signaling pathway.

This study was aimed to clone buffalo SIRT6 gene,construct retroviral expression vector,and conduct bioinformatics and tissue expression profile analysis.Primers were designed by Oligo 7.0 according the coding sequence(CDS) of cattle SIRT6 gene(GenBank accession No.:NM_001098084.1),the SIRT6 gene CDS was amplified by RT-PCR,and analyzed by bioinformatic after sequencing.The recombinant retroviral vector pMXs-SIRT6-IRES-GFP was constructed,and expressed in HEK-293T cells and buffalo fetal fibroblasts (BFF).RNA was extracted from heart,liver,spleen,lung,kidney,intestine,brain,skin and genital ridge in buffalo,respectively,the relative expression of SIRT6 gene in different tissues was detected by Real-time quantitative PCR using cDNA as template.The results showed that the coding region of SIRT6 gene in buffalo was 1 080 bp in length and encoded 359 amino acids,the contents of leucine and proline were the highest (10.3%) and tyrosine was the lowest (1.1%).The homology of nucleotide sequence of SIRT6 gene in buffalo were 98.4%,86.7% and 78.5% with Bos taurus,Homo sapiens and Mus musculus,respectively,the homology among species was low.The phylogenetic tree results showed that buffalo and Bos taurus were clustered into one branch,and then Homo sapiens and Mus musculus were clustered into one large branch,the relationship was relatively close,but far from that of Drosophila melanogaster.The theoretical molecular weight of SIRT6 protein was 39.47 ku,the molecular formula was C₁₇₃₇H₂₇₈₃N₅₀₉O₅₁₆S₁₃,and the isoelectric point was 8.48,there was no transmembrane region,and it was an intramembrane protein.SIRT6 protein contained the catalytic structure of Sirtuin family-specific SIRT2 deacetylase.Secondary structure prediction showed that buffalo SIRT6 protein contained 13 α-helix,27 β-sheet,24 T-turn and 20 random coil.The retroviral vector mediated SIRT6 gene could be expressed in HEK-293T cells and BFF.Real-time quantitative results showed that SIRT6 gene was expressed in heart,liver,spleen,lung,kidney,intestine,brain,skin and genital ridge of buffalo,and the expression of SIRT6 gene was the highest in genital ridge,followed by in skin,and was the lowest in liver.This results would lay a foundation for the functional study of SIRT6 gene in buffalo.

To screen the enhancer element of regulation Tβ4 gene transcription in Min pig,and explore the expression regulation mechanism of it,in this study,a series of truncated fragments of the Tβ4 gene promoter region were amplified by PCR using the genomic DNA in Min pig.PCR products were ligated into pMD18-T vector,the cloning plasmid was constructed.The cloning plasmid was ligated into pGL3-basic vector by double digestion and ligation reaction,the relative luciferase activity of this recombinant plasmid was determined in PK15 cell by dual luciferase detection system.The core region of Tβ4 promoter was further screened according to the number of relative luciferase activity.At the same time,three online softwares were used to forecast the binding site of transcription factor in promoter core region.Based on the forecast results,an overlapping PCR was used to site-directed delete in transcription factor binding site,the mutant vectors were constructed using these PCR products and transfected into PK15 cell using wild type plasmid as control.The results showed that six different lengths of Tβ4 gene promoter fragments were successfully constructed,five of them had significant activity.After two rounds of double luciferase activity assay,-155 to -105 bp region was determined as the core region of Tβ4 promoter in Min pig,through bioinformatics analysis,there were three transcription factor binding sites in this region,they were E2F-1,MYBAS1 and ELK-1.Three mutant vectors with deletion of transcription factors were constructed by site-directed deletion,only the deletion of ELK-1 binding site was found by double luciferase assay,which would result in a significant decrease in promoter activity (P<0.05).It was speculated that ELK-1 was a positive regulatory element of transcription of Tβ4 gene in Min pig.

The aim of this study was to investigate the expression characteristics of cyclic AMP-responsive elmment-binding protein 3-like 3 (CREB-H) in different tissues of pig and the expression patterns of CREB-H in liver of Mashen and Large White pigs.Real-time quantitative PCR and Western blotting were used to detect the expression profile of CREB-H gene in 12 tissues (heart,liver,spleen,lung,kidney,stomach,small intestine,cerebellum,hypothalamus,longissimus dorsi muscle,biceps femoris and psoas major) of Mashen pig and the expression patterns of CREB-H in liver of Mashen and Large White pigs at 1,30,60,90,120,150 and 180 days of age.The results showed that CREB-H gene mRNA was widely expressed in 12 tissues of sampled pigs,and the expression was high in liver and small intestine.The expression of CREB-H protein in liver was significantly higher than that in other tissues (P<0.05),and it was not expressed in heart,spleen and cerebellum.The developmental expression patterns of CREB-H gene mRNA and protein occurring in liver of pigs were affected by days of age,breed and the interaction of breed and days of age (P<0.01).The expression of CREB-H gene mRNA and protein in liver were the highest at 1 day of age after birth between two breeds,and the expression of CREB-H protein in Mashen pig was extremely significantly higher than that in Large White pig at each stage (P<0.01),and CREB-H was mainly expressed in liver of pig.There were temporal and spatial differences of the expression in liver between Mashen and Large White pigs,which might be relate to lipid metabolism in pigs at different developmental stages,this results provided a theoretical basis for studying the regulation mechanism of lipid metabolism in pigs.

The aim of this study was to investigate the potential role of sex hormone-binding globulin (SHBG) in regulating yak reproduction by clarifying its CDS sequence and expression characteristics in the reproductive axis of female yak (Bos grunniens).Tissue samples of hypothalamus,anterior pituitary,oviduct,ovary and uterus were collected from five adult and health female yaks and cattle,respectively.The SHBG gene-specific amplification primers of yak were designed according to bovine SHBG gene in NCBI.Cloning,bioinformatics analysis and tissue expression of yak SHBG gene were performed by RT-PCR,bioinformatics methods and Real-time PCR,respectively.The result showed that the coding region (CDS) of yak SHBG was 1 206 bp,encoding a 401 amino acids protein which contained a high proportion of leucine (16.5%),glycine (9.7%),proline (8.7%) and serine (9.0%).The formula of SHBG protein was C₁₉₄₄H₃₀₆₁N₅₃₅O₅₆₆S₁₀ and its molecular weight and theoretical isoelectric point were 43.30 ku and 5.63,respectively.Yak SHBG had the highest homology with cattle in nucleotide sequence (99.0%) and amino acid sequence (97.5%).It was a hydrophilicity instability protein contained a signal peptide and a transmembrane region.Random coil and extend chain were mainly in the secondary structure of SHBG,which was consistent with the result of the tertiary structure analysis.The phylogenetic tree showed the closest genetic relationship between yak and cattle.The expression of SHBG gene in yak oviduct was significantly higher than that in other tissues (P<0.05),and the expression among other tissues was not significantly different (P>0.05).The expression of SHBG gene in cattle oviduct was extremely significantly higher than that in yak (P<0.01),and the expression in cattle ovary was significantly higher than that in yak (P<0.05).The expression of SHBG gene in other tissues was not significantly different between yaks and cattle (P>0.05).The research on the SHBG gene sequence and its expression in the reproductive axis of the yak provided a theoretical basis for further studying on the regulation of SHBG on the reproduction in yak.

Sirt3,a member of the Sirtuin family,has a high degree of deacetylase activity,which regulates the growth,aging,metabolism and other physiological processes of animals.Current research indicates that Sirt3 can activate superoxide dismutase,regulate tricarboxylic acid cycle and promote electron transport chain to eliminate intracellular reactive oxygen species (ROS),avoid oxidative damage of cells and damage of mitochondrial membrane potential,to affects apoptosis and autophagy;Sirt3 can slow down aging by regulating ROS and reducing the incidence of various degenerative diseases.In addition,the Sirt3 affects muscle development by various ways such as activating AMP-dependent protein kinase (AMPK).Studies on pigs showed that there were differences in meat quality such as muscle color and water loss rate of different Sirt3 mutants,and Sirt3 had an effect on fatty acid oxidation and ketone body formation.The study of the cattle Sirt3 protein,which has high homology to the pig Sirt3 protein,found that there were significant differences in the body length,body height and muscle fat content of different mutant Sirt3.The authors summarized the regulation mechanism of Sirt3 on mitochondrial energy metabolism and oxidative damage,and its effects on apoptosis,autophagy and aging.They also described partial physicochemical properties of Sirt3 protein of pig and the mechanism of its effects on pig growth and development,in order to explore the biological role of this gene in pig muscle and fat development and provide a theoretical basis for animal production improvement.

Under normal physiological conditions,the generation and scavenging of free radicals in animals are in a stable dynamic balance.However,when oxidative stress occurs,the amount of free radicals in the body exceeds the scavenging capacity,causing the imbalance between the oxidation system and the antioxidant system and causing oxidative damage.Activation of the Keap1-Nrf2/ARE signaling pathway plays a key role in preventing cellular and tissue damage caused by oxidative stress,maintaining redox balance and protein homeostasis,and regulating inflammation.Keap1 is a cytoplasmic inhibitor of Nrf2.Under normal physiological conditions,Nrf2 binds to its inhibitor Keap1 to undergo ubiquitination and proteasomal degradation,maintaining the Nrf2 content in the nucleus at a relatively low level.Under oxidative stress,the ability of Keap1 to ubiquitinate and degrade Nrf2 is weakened,Nrf2 accumulates in the nucleus,and Nrf2 binds to the antioxidant response element (ARE) to initiate the expression of a series of downstream protective genes,which makes the body returns to a normal physiological state from oxidative stress.Oxidative stress is involved in a variety of pathophysiological changes,which have a negative impact on livestock production performance,growth and development,reproductive performance,meat quality,etc.Keap1-Nrf2/ARE signaling pathway is one of the most important antioxidant stress pathways in cells,so it is extremely important to the health protection of animals.The authors summarizes the basic structure and activation mechanism of Keap1-Nrf2/ARE signaling pathway and the research progress in livestock and poultry,and describes its important role in improving the antioxidant,anti-inflammatory and meat quality of livestock and poultry,in order to provide a theoretical basis for solving the serious oxidative stress and inflammation in the current livestock industry.

The purpose of this experiment was to study the effect of estrogen on apoptosis and growth cycle of bovine mammary epithelial cells (BMECs).The specific mechanism of estrogen regulating apoptosis and growth cycle of BMECs was explored by adding MAPK/ERK signaling pathway blockers.Flow cytometry was used to detect apoptosis and cycle changes.Real-time PCR was used to detect the expression abundance of Bcl-2,Caspase3 and CyclinD1 genes.The results showed that the apoptotic rate of BMECs in control group was extremely significantly lower than that in BMECs+PD98059 and BMECs+E₂+PD98059 groups (P<0.01),the expression abundance of Bcl-2 was extremely significantly higher than that in BMECs+PD98059 group (P<0.01),and the expression abundance of Caspase3 was significantly lower than that in BMECs+PD98059 group (P<0.01);In control group,the proportion of G1 phase cells was significantly higher than that in BMECs+E₂ group (P<0.05),and was extremely significantly lower than that in BMECs+E₂+PD98059 group (P<0.01),the proportion of S phase cells was significantly higher than that in BMECs+PD98059 and BMECs+E₂+PD98059 groups (P<0.01),the proportion of G2 phase cells was extremely significantly lower than that of BMECs+PD98059 and BMECs+E₂+PD98059 groups (P<0.01);The expression abundance of CyclinD1 gene was extremely significantly higher than that in BMECs+PD98059 group (P<0.01);The expression of Bcl-2 gene in BMECs+E₂+PD98059 group were extremely significantly higher than that in BMECs+PD98059 group (P<0.01),and the expression of Caspase3 was extremely significantly lower in BMECs+PD98059 group than that in BMECs+PD98059 group (P<0.01).The results showed that the MAPK/ERK signaling pathway was involved in the regulation of proliferation and cell growth cycle of mammary epithelial cells,and estrogen could inhibit the apoptosis of mammary epithelial cells through the MAPK/ERK signaling pathway.The MAPK/ERK signaling pathway might be involved in the process of cell growth cycle regulated by estrogen.

This experiment was aimed to study the effects of Monascus synbiotic,yeast-selenium-germanium culture and their compound preparation instead of antibiotics on growth performance of weaned piglets.Eighty healthy piglets (7.57 kg±0.49 kg) with (28±1) d were randomly divided into 4 groups with 5 replicates per group and 4 piglets per replicate.The pigs in control group were fed basal diet with 25 mg/kg colistin sulfate and 45 mg/kg chlortetracycline.The diets of groups 1,2 and 3 were added with 0.5% Monascus synbiotics,0.25% Monascus synbiotics+0.25% yeast-selenium-germanium culture and 0.5% yeast-selenium-germanium culture.The test period was 42 d,which was divided into 3 stages,14 days per stage (1-14,15-28,29-42 d).The results showed that:①Compared with the control group,in all stages of the experiment,the ADG of the groups 1 and 2 were significant or extremely significant increased (P<0.05;P<0.01).At 29-42 d,the F/G of all the experimental groups were significantly decreased (P<0.05).Compared with the control group,the final weight of piglets in the experimental groups had extremely significant or significant increased (P<0.01;P<0.05).②Compared with the control group,the diarrhea rate (DR) and diarrhea index (DI) of the group 1 were significantly decreased (P<0.05) on the 1-7 d of the experiment.In the whole trial period,compared with the control group,the DR and DI of the group 1 and the DR of the group 2 were significantly lower (P<0.05).Compared with the group 3,the DI of the group 1 and 2 showed an extremely significant or significant decrease (P<0.01;P<0.05).③Compared with the control group,the apparent digestibility of CP and EE of the groups 1 and 2 were significantly or extremely significantly increased (P<0.05;P<0.01) at the 1-28 d of the experiment.Compared with the control group,the apparent digestibility of DM,CP and EE of all the experimental groups were significantly or extremely significantly increased (P<0.05;P<0.01),and the apparent digestibility of P of the groups 1 and 2 were significantly increased (P<0.05) on the 29-42 d of the experiment.④Compared with the control group,the serum GH and IGF-1 levels of all the experimental groups were significantly or extremely significantly increased (P<0.05;P<0.01),and the serum GHRIH of all the experimental groups was significantly or extremely significantly decreased (P<0.05;P<0.01).Among them,the serum IGF-1 level of the group 2 was significantly higher than that in the groups 1 and 3 (P<0.05).Compared with the control group,the serum COR levels of the groups 1 and 2 were significantly increased (P<0.05).In summary,compared with antibiotics,0.5% compound preparation of Monascus synbiotic and yeast-selenium-germanium culture could effectively prevent diarrhea in weaned piglets and improve the growth of weaned piglets.The 0.5% Compound Preparation of Monascus synbiotic and yeast-selenium-germanium culture could replace 25 mg/kg colistin sulfate and 45 mg/kg aureomycin in diet under this experiment.

The experiment was conducted to study the effect of different storage temperature and time on freshness,composition,egg quality and albumen transparency of pigeon eggs,in order to discuss optimal storage condition of pigeon eggs,and explore the change rule of pigeon albumen transparency under different storage conditions,finally provide the theoretical basis for reasonable storage and sale of pigeon egg.1-2 year old White King pigeons with male-female pairs were chosen as the experimental object,3 (low temperature,middle temperature,high temperature)×4 (1w,2w,3w,4w) was designed.90 pigeon eggs were randomly collected each week,and were placed under three different temperatures for 1,2,3 and 4 weeks respectively,30 eggs each group.The freshness,composition,egg quality and albumen transparency of pigeon eggs were determined.The results showed that there was no scattered yolk or mildew after storing for 4w under low temperature,but the pigeon eggs could only be stored for 1w without deterioration under middle and high temperature.As time went on and the temperature went up,the egg weight loss rate showed a significant upward trend (P<0.05).The storage time had a significant effect on the eggshell rate,the albumen rate and the yolk rate (P<0.05),but the storage temperature had no effect on the three abrove indexes (P>0.05).The storage time and temperature had significant effects on the albumen height and yolk color (P<0.05).The albumen height showed the downward trend,and the yolk color darkened as time went on and the temperature went up.There were significant effects of the storage time and temperature on L^*,a^* and b^* values of cooked pigeon albumen (P<0.05).The percentage of transparent pigeon eggs was the highest under middle temperature.In conclusion,pigeon eggs should be stored and sold under refrigeration,and pigeon eggs could be stored for more than 4 w in cold storage condition.The albumen transparency of pigeon eggs could be improved under middle temperature (about 20℃).

Mycotoxins are secondary metabolites produced by different fungi,which have significant toxic effects on animals and humans,including reducing livestock and poultry production performance,damaging organ function,affecting the immune function of the body,causing oxidative damage to cells and DNA,and even leading to animal death.Mycotoxin contamination of feed and raw materials is a widespread,global problem that can occur both in crop field and during storage.Based on this,each year,the contamination of the feed and its raw materials has caused huge economic losses to the livestock and poultry breeding industry.Therefore,prevention and control of mold growth and removal of mycotoxins in feed has become a hot research topic at home and abroad.Indeed,different mycotoxins have different adverse effect on different animals,and the addition of appropriate substances (montmorillonite and enzyme preparations) during livestock and poultry breeding can effectively inhibit the mycotoxin activity and alleviate the toxicity of mycotoxins.In this paper,the harm of zearalenone,fumonisins,deoxynivalenol,T-2 mycotoxin,aflatoxins and ochratoxins to livestock and poultry were systematically discussed and the methods of reducing mycotoxins in feed and its raw materials from the physical,chemical and biological perspectives were introduced,so as to provide a basis for the rational and effective prevention and control of mycotoxins contamination and the protection of healthy breeding of livestock and poultry.

This study was conducted to investigate the effects of VE-riched transgenic maize on growth performance,biochemical parameters and antioxidant capacity of broilers,and evaluate the nutritional effectiveness of VE-riched transgenic maize compared with the non-genetically modified maize (non-GM maize) added exogenous DL-α-tocopherol acetate additive.360 1-day-old Ross 308 male broilers were randomly divided into 3 groups with 6 replicates and 20 chickens in per replicate.The study period was 42 d.The diet for ordinary maize negative control group (negative control group,NC) was prepared with non-GM maize without exogenous VE and the VE level of diets during 1 to 21 d and 22 to 42 d were 4.38 and 4.63 IU/kg,respectively;Diet for VE-riched transgenic maize group (VE-riched group,VER) was prepared with VE-riched transgenic maize and the VE level of diets during 1 to 21 d and 22 to 42 d were 14.11 and 14.91 IU/kg,respectively;Diet for ordinary maize positive control group (positive control group,PC) was prepared with non-GM maize added DL-α-tocopherol acetate additive to make VE level consistent with VE-riched group.The results showed as follows:①Except for average daily feed intake (ADFI) of 1 to 21 d in VE-riched group which was significantly lower than that in negative control group (P<0.05),there was no significant difference in other growth performance parameters among the three groups during the experiment (P>0.05).②On 21 d,the serum ALT of VE-riched group was not significantly different from positive control group (P>0.05),but significantly lower than that of negative control group (P<0.05),and there was no significant difference in liver TG content between VE-riched group and negative control group (P>0.05),and the two groups were significantly higher than that of positive control group (P<0.05);On 42 d,there was no significant difference in the liver TG content between VE-riched group and positive control group (P>0.05),and the two groups were significantly lower than that of negative control group (P<0.05);③Compared with positive control group,serum MDA,H₂O₂,GSH,GSSG,SOD,CAT and T-AOC of 21 d and serum MDA,H₂O₂,GSH,SOD,CAT and T-AOC of 42 d in VE-riched group had no significant difference (P>0.05);Compared with negative control group,serum MDA and H₂O₂ of 21 d and 42 d significantly decreased in VE-riched group (P<0.05),meanwhile,GSH,GSSG,GSH-Px,SOD,CAT and T-AOC of 21 d and GSSG,GSH-Px,CAT and T-AOC of 42 d increased significantly in the VE-riched group (P<0.05).The above results demonstrated that VE-riched transgenic maize and non-GM maize added with exogenous DL-α-tocopherol acetate additive had similar effects on growth performance,biochemical parameters and antioxidant capacity of the test broilers under the same VE level.

The early stage is a very important stage in the life of ruminants.The nutrition supply,growth and development status of the ruminant during this period determine the production performance and reproductive efficiency in adulthood.Due to its immature digestive and metabolic system and extremely strong plasticity in the young stage,the dietary nutrition levels of ruminants,especially the protein and neutral detergent fiber (NDF) levels,play a very important role in the development process of ruminants.In production practice,inadequate or excessive nutrition supply will affect the growth and health of young animals,and even accompany them throughout their life.The dietary protein and fiber levels of young ruminants play a decisive role in the regulation of growth performance and gastrointestinal development.An appropriate increase in dietary protein level can improve the growth and development of young ruminants,and improve nutrient digestibility and rumen development due to the increase in available nitrogen concentration of rumen microorganisms in the feed.Under the condition of ensuring sufficient concentrate feed,increasing the dietary fiber level can improve the feed intake and growth performance of young ruminants,and promote rumen development,as well as improve the digestibility of nutrients.However,due to the different sources of experimental animals or raw materials,there are still different experimental results in related experiments.Therefore,the authors focus on the effects of different levels of dietary protein and NDF on the growth and development,nutrient digestibility and rumen development in young ruminant,and the reasons for different experimental results,in order to provide theoretical basis for solving the problem on the breeding management of young ruminant animal,and improving their feeding efficiency.

This study was aimed to investigate the polymorphism of AT-rich interactive domain 5B (ARID5B) gene and its correlation with the body size and meat quality traits in Yanhuang cattle.99 Yanhuang cattle at 18 months old were selected as the research object,the SNP of 13 exons of ARID5B gene was found using PCR sequencing,the polymorphism of SNP was analyzed by HRM typing,and the association between ARID5B gene polymorphism and the body size and meat quality traits in Yanhuang cattle was analyzed by SPSS 19.0 software.The results showed that A/G mutation was detected at Chr28:18186635 bp of exon 10 of ARID5B gene in Yanhuang cattle,which contained three genotypes:AA,AG and GG,AA genotype was the dominant genotype,and A was the dominant allele.The chi-square suitability test result showed that Yanhuang cattle population was in Hardy-Weinberg equilibrium (P>0.05),population genetic parameter analysis result showed that the heterozygosity of the mutation site was relatively low,and had a low varitation in Yanhuang cattle population;Polymorphism information content (PIC) analysis showed that it was a moderately polymorphic (0.25 < PIC < 0.5).The correlation analysis results showed that there was a significant difference between the GG genotype of exon 10 of ARID5B gene and the body height,chest circumference,abdominal circumference and intramuscular fat content in Yanhuang cattle (P<0.05).This experiment revealed the correlation between ARID5B gene and meat traits in cattle,which provided a reference for exploring the feasibility as an effective genetic marker,and provided experimental basis for molecular breeding of Yanhuang cattle.

This study was aimed to analyze the genetic diversity and phylogenetic evolution in Xianglushan chicken.The full-length sequences of mitochondrial DNA D-Loop (mtDNA D-Loop) region of 121 Xianglushan chickens were analyzed by PCR amplification combined with bidirectional sequencing,and the composition,variation and maternal origin of mtDNA D-Loop region were discussed.The results showed that the total length of mtDNA D-Loop region in Xianglushan chicken was 1 231-1 233 bp.The contents of A,T,C and G were 26.62%,33.55%,26.49% and 13.34%,respectively.The content of T was the highest,the content of G was the lowest,and the content of A+T was significantly higher than that of G+C,it indicated that region might have certain base hobbies.Analysis of 121 full-length sequences were found to coexist in 11 haplotypes and 26 mutation sites,of which 2 were single polymorphic information sites,24 were simple information sites,4 bases were inserted and 2 bases were missing.The genetic diversity analysis results showed that the haplotype diversity was 0.814,the nucleotide diversity was 0.00447,the average nucleic acid difference was 5.494,which indicated that the genetic diversity of Xianglushan chicken was relatively rich,the effect of preservation was better,which had a certain breeding space.Tajima's D was 0.39378 and the test results were not significant (P>0.10),in line with neutral mutations.The cluster analysis results showed that Xianglushan chicken and Gallus gallus gathered as one,indicating that Xianglushan chicken originated from Gallus gallus, and there were 4 branches inside the branch,indicating that Xianglushan chicken had many matrilineal origins.The results could provide some reference data for the protection and exploitation of pheasant germplasm resources in Xianglushan chicken.

This study was aimed to explore the association between the polymorphism of stearoy-CoA desaturase 1 (SCD1) gene and economic traits in Yanbian Yellow cattle.157 healthy 36-month-old Yanbian Yellow cattle of similar weight were selected,and the blood genomic DNA were extracted from the jugular vein.According to the sequence of bovine SCD1 gene in GenBank (accession No.:AC_000181.1),specific primers were designed by Oligo 6.0 software.The full-length SCD1 gene was amplified by PCR and sequenced directly,the polymorphic was analyzed,and the protein structure was analyzed by bioinformatics software.The carcass traits such as live weight,carcass weight,slaughter rate and backfat thickness were measured before slaughter,the longissimus dorsi muscle was collected and the meat traits such as eye muscle area,cooking loss,drip loss,tenderness,hydraulic power,pH and fatty acid content were measured after slaughter.The correlation between the polymorphism of SCD1 gene and meat quality traits in Yanbian Yellow cattle was analyzed by general linear model analysis.The results showed that there were A→G mutation at 846 bp (g846 A→G) and C→T mutation at 1 022 bp of SCD1 gene (g1022 C→T).g1022 C→T mutation resulted in the mutation of alanie (A) to valine (V),but did not cause tertiary structural changes.Correlation analysis results showed that there was a significant correlation between g846 A→G site and the live weight,carcass weight,drip loss,and the content of stearic acid and linoleic acid (P<0.05),and there was a significant correlation between g1022 C→T site and the live weight,carcass weight and stearic acid content (P<0.05).The results indicated that the two polymorphic sites were significantly correlated with the meat quality traits of Yanbian Yellow cattle,which could be used as a potential molecular marker.

Sperm mitochondria is the key organelles to maintain sperm motility for mammals,which play important roles in regulating sperm hyperactivation,capacitation,acrosome reaction and fertilization.The unique morphological characteristics and specific isomer enzyme of mammalian sperm mitochondria give them unique kinetics and regulation properties.The oxidative phosphorylation in sperm mitochondria is an important way to maintain sperm motility,and a proper amount of reactive oxygen produced during this process is crucial for maintaining sperm function,however,excessive level may lead to sperm damage and accelerate sperm apoptosis.Mammalian sperm plasma membrane phosphatidylserine valgus and related caspase activation cascades cause apoptosis.Interestingly,unlike somatic mitochondria,mitochondrial calcium signaling may not be involved in the intrinsic apoptosis pathway of sperm.Increasing studies focus on mitochondrial membrane potential and oxygen consumption which are believed to be sensitive indicators to evaluate mammalian sperm mitochondrial functions.Mammalian sperm mitochondria have their own genetic systems,and mitochondrial gene copy numbers may serve as markers for non-invasive measurement of sperm quality and fertility.This paper mainly reviews the structure of mammalian sperm mitochondria,the formation of mitochondrial sheath,and the relevant biological functions,including the oxidative phosphorylation process in mitochondria,the advantages and disadvantages of reactive oxygen species on sperm,and the involvement of mitochondria in calcium homeostasis and cell apoptosis.Apart from that,the studies related to mitochondrial membrane potential detection,oxygen consumption and mitochondrial genome were summarized,which provided a basis for further exploration of sperm motility mechanisms involved in mitochondria.

The aim of this study was to investigate the genetic polymorphism of MDHⅠ gene and its associations analysis with growth traits,in order to provide a reference for the breeding of qualify traits in yak.Five groups (178 total) of yak were selected,DNA were extracted from the ear samples,and DNA pools were constructed.The specific primers of all exons of MDHⅠ gene were designed for amplification and sequencing.SNPs were screened using Mega 5.2,the genotypes were identified by direct sequencing.χ² independence detection,gene homozygosity (Ho),heterozygosity (He),effective allele frequency (Ne) and polymorphism information the content (PIC) analysis were analyzed by PopGene 32,the association between the polymorphisms of MDHⅠ gene and growth traits in yak was analyzed using SPSS 24.0.The results showed that only one mutation site in exon 8 (G23093C,synonymous mutation) was found in Shenzha,Leiwuqi,Sibu,Pali and Maiwa yaks,which was synonymous mutations.There was three genotypes of GC,CC and GG,among the five yak populations,GC genotype was the dominant genotype (GG was the dominant genotype in Leiwuqi yak),and G was the dominant allele (except for Sibu yak),the gene frequency of G and C were equal in Sibu yak.χ² adaptability test showed that Shenzha,Leiwuqi,Sibu,Pali and Maiwa yaks were in accordance with Hardy-Weinberg equilibrium (P>0.05).MDHⅠ gene in Shenzha,Pali and Sibu yaks showed highly polymorphic (PIC>0.5),while Maiwa and Leiwuqi yaks showed moderately polymorphic (0.25 < PIC < 0.5),and the highest homozygosity was Shenzha and Leiwuqi yaks.The correlation analysis results showed that different genotypes of MDHⅠgene had significant effects on body weight in Leiwuqi,Pali and Maiwa yaks (P<0.05),and the interspecies analysis results showed that different genotypes of MDHⅠ gene had significant effects on body weight in yak (P<0.05).It indicated that MDHⅠ gene could be used as a molecular marker for artificial breeding.

Animal coat color is an easily recognized phenotypes,and also be used as an effective measures of screening for some diseases.Coat color is mainly determined by the distribution,proportion and production rate of eumelanin and pheomelanin which were produced by melanocytes.Many genes play an important regulatory role in the production and distribution of melanin,and different genotypes of each gene form a variety of basic coat color,fade coat color and spot.In addition,miRNA binds the mRNA of coat color major gene to induce inhibition/enhancement of its transcription and translation process,thereby regulating the expression of coat color gene and affecting the synthesis of melanin.In this paper,the relationship between the coat color traits,diseases and DNA sequence polymorphisms (different genotypes) of major candidate genes MC1R,ASIP,KIT,TYRP,MITF,EDNRB in livestock were briefly described.The research progress on coat color formation miRNA mining and identification,miRNA regulation of major coat color genes,and the application value of coat color gene were analyzed which would provide a theoretical basis for future study on coat color mechanism of livestock.

Litter size is a significant economic trait in pig industry,and embryo implantation plays an important role in the litter size of pigs.Regulation of embryo implantation involves many biological processes,in which there are many substances (implantation factors) play functions.In this study,the authors summarized the relevant researches on progesterone,progesterone receptor,estradiol,estrogen receptor α (ESR1) and erythropoietin-producing hepatocellular receptor-ligand (Eph-ephrin) system,and the paper is focused on the spatial and temporal expression,function,mutation and regulation of these factors during porcine embryo implantation.Among these implantation factors,progesterone and its receptor play an important role in the whole process of embryo implantation.Progesterone secreted by the corpus luteum combines with the progesterone receptors in the tissues of endometrial epithelium,glandular epithelium,stroma and muscle in the pregnant sows,and the combination stimulates cells of these tissues to secrete a variety of implantation factors,which are further involved in the embryo implantation process.Estradiol is one of the most abundant and active estrogens and it is mainly secreted by ovarian granulosa cells,and ESR1 is the main receptor of estradiol in uterus.Combination of estradiol and ESR1 can make the sow estrus.In addition,blastocyst in the free state also secretes estradiol,which is a signal that is allowed to be recognized by endometrium of the sow.Eph-ephrin system has an extensive function,including its role in regulation of embryo implantation in human,mouse and pigs.EphA1,ephrin A1 and EphA4 genes of Eph-ephrin system have significantly higher expression in pregnant pigs during embryo implantation than that of non-pregnant pigs,and expression of these genes in endometrial attachment sites are significantly higher than that of inter-attachment sites.Besides,inhibition of ephrin A1 could reduce the migration and adhesion ability of endometrial epithelial cells.These implantation factors play important roles in porcine embryo implantation activities.In-depth researches on these factors will help to clarify the regulation mechanism of embryo attachment and further reveal the mechanism of high litter size in pigs.

In order to understand the genetic variation and molecular characteristics of avian leukaemia virus (ALV) endemic strains in Guizhou,this study designed and synthesized primers based on ALV env gene for the purpose of gene amplification,cloning and sequence analysis of avian leukaemia clinical cases.The results showed that 3 positive samples were selected from clinical cases.The target gene fragments with a size of 921 bp were obtained by PCR amplification and named as GZ-ALV-1,GZ-ALV-2 and GZ-ALV-3 strains.Sequence analysis results showed that the nucleotide homology of 3 ALV epidemic strains in Guizhou ranged from 97.2% to 97.6%,which was relatively high with ALV-J at home and abroad,ranging from 93.1% to 99.3%,while the homology with ALV subgroups A,B,C,D,E and K was only 51.4% to 53.2%.The phylogenetic tree analysis showed that 3 ALV epidemic strains in Guizhou were in the same branch as the reference strains of ALV-J subgroup,which further indicated that all the ALV strains detected in this paper were ALV-J subgroup,while in different branches compared with A,B,C,D,E and K subgroups.The gene mutation analysis showed that 37 same nucleotide mutations of 3 epidemic strains resulted in 17 amino acid mutation sites,including 9 variable points in the high-variable region of hr1 and hr2,and 1 variable point in the low-variable region of vr3.The results showed that 3 ALV strains in Guizhou were ALV-J subgroup,and env gene loci were mutated,and the variable loci were located in the high-variable region.The results provided basic data for the epidemic situation of avian leukemia and the prevention and control and purification of ALV in Guizhou province.

This experiment was aimed to establish an immunoperoxidase monolayer assay (IPMA) screening method for the detection of anti-H1N1 subtype swine influenza virus monoclonal antibody.The specificity,sensitivity and reproducibility of the established IPMA screening method were evaluated by optimizing the reaction conditions of MDCK cells,culture time after cell inoculation,type of blocking solution,working concentration and working time.The results showed that the optimal reaction conditions for the established IPMA were MDCK cells with 10^2.63 TCID₅₀/100 μL H1N1 subtype swine influenza virus,cultured at 37℃ for 24 h,3 ‰ H₂O₂ methanol fixed at room temperature for 15 min,5% skim milk blocking for 2 h at 37℃,50 μL of hybridoma cell supernatant was used as primary antibody,incubated at 37℃ for 2 h,and goat anti-mouse HRP-IgG secondary antibody was incubated at 37℃ for 1 h.The established IPMA method could specifically detect H1N1 subtype swine influenza virus monoclonal antibodies,and did not cross-react with porcine reproductive and respiratory syndrome virus (PRRSV),porcine circovirus type 2 (PCV2) and swine fever virus (CSFV) positive serum.The sensitivity test results showed that 1:3 200 HI=2^-9 standard H1N1 swine positive serum could be detected;The repeatability test between inter-batch and intra-batch was good.In summary,this experiment successfully established an IPMA detection method for anti-H1N1 subtype swine influenza virus monoclonal antibody,which had strong specificity,high sensitivity and good reproducibility,and provided a simple,practical and effective detection method for the production of monoclonal antibodies against H1N1 subtype swine influenza virus.

In order to explore the technique of regulating the reproductive performance of domestic geese,this study designed a synthetic goose gonadotropin-inhibitory hormone (GnIH) sequence fragment,which was digested and ligated to Norovirus (Nov) P gene recombinant pEGX-4T-1 vector,transformed into E.coli DH5α competent cells,extracted positive bacterial recombinant plasmid,and verified by sequencing and enzyme digestion.The correctly sequenced plasmid was transformed into E.coli BL21 competent cells,and recombinant protein expression was induced by IPTG.The expression of recombinant protein was detected by SDS-PAGE and Western blotting.The prokaryotic expression was further carried out under different IPTG concentration and induction time conditions,and the optimal induction conditions were determined.Under the optimal conditions,the induced bacterial liquid was collected by centrifugation,and the protein was analyzed by ultrasonic disruption and centrifugation.The recombinant protein was purified using xTractor Buffer and Glutathione Superflow resin,and the recombinant protein was detected by Western blotting using self-made anti-GnIH goose serum and anti-goose secondary antibody.The results showed that the recombinant plasmid Nov P-partical-GnIH was identified by double restriction enzyme digestion to obtain the target gene band of about 96 bp.The sequencing results confirmed that the recombinant plasmid was successfully constructed.The molecular weight of the recombinant protein induced by expression was about 64 ku,which was consistent with the expected size,and the highest expression was obtained at 37℃,1.0 mmol/L IPTG and induction for 2 h.The recombinant protein was detected by SDS-PAGE after sonication.Mainly expressed in the precipitate.The recombinant protein reacted with both GST monoclonal antibody and anti-GnIH positive serum,indicating that the recombinant protein had good reactogenicity and immunogenicity.In this study,a large number of highly purified recombinant proteins were successfully obtained by prokaryotic expression and purification of Nov P-partical-GnIH protein,which would help to further establish the reproductive regulation of domestic geese and lay the foundation for studing on improving reproductive performance and regulation reproduction mechanism.

The aim of the experiment was to express and purify porcine NLRP6 protein and prepare mouse anti-NLRP6 polyclonal antibody.Specific primers were designed according to the nucleotide sequence of porcine NLRP6 (GenBank accession No.:XM_003124236.4).NLRP6 gene fragment was amplified from pMD-19T-NLRP6 recombinant vector by PCR method,and the amplified product was connected with prokaryotic expression vector pET-32a(+) to obtain recombinant plasmid pET-32a-NLRP6.After identification,PCR and sequencing analysis,it was transformed into competent cells of Escherichia coli Rosetta(DE3),and expressed by IPTG and identified by Western blotting.The recombinant fusion protein was purified by denaturation,nickel affinity purification and renaturation.The recombinant fusion protein was immunized to BALB/c mice to prepare polyclonal antibodies.The results showed that a 576 bp NLPR gene was successfully cloned,and the recombinant plasmid pET-32a-NLRP6 was constructed correctly.The recombinant fusion protein of NLRP6 with a size of about 34 ku was obtained by IPTG induction.Western blotting analysis showed that the recombinant protein was positive for mouse anti-6×His monoclonal antibody.The solubility analysis showed that the recombinant fusion protein of NLRP6 existed in the form of inclusion bodies,accounting for about 95%.The purified NLRP6 recombinant fusion protein was used to immunize BALB/c mice to obtain polyclonal antibodies.Western blotting analysis showed specific reactions.The immunogenic NLRP6 protein and its mouse-derived polyclonal antibodies were successfully prepared in this experiment,which provided basic materials for the study of biological functions of NLRP6 protein and pathogenesis of related diseases.

To understand the genotype of extend spectrum β-lactamase (ESBLs) and AmpC β-lactamase(AmpC) of Escherichia coli isolated from chicken and actuality of antibiotics resistance in Shanxi province,68 isolated strains accorded with biological traits of chicken Escherichia coli were identified from epidemic materials possessed typical clinical symptoms of chicken colibacillosis.These isolated strains were subjected to K-B disc diffusion and double-disc synergy confirmatory methods recommended by CLSI for drug sensitivity test and resistant phenotype;Genotype of ESBLs and AmpC were detected by PCR method.The results showed that all strains presented resistance to 22 antibiotics in different degrees.The resistant spectrum of 66 strains,accounting for 97.06%,reached to above seven kinds of antibiotics;The number of ESBLs-positive,AmpC-positive and double positive strains were 57(83.82%),19(27.94%) and 16(23.53%),respectively;Resistant genotypes of ESBLs-positive strains were bla_TEM,bla_OXA and bla_CTX-M-1/9,and the genotype of AmpC-positive strains was bla_FOX.The results of this research indicated that all isolated strains presented resistance to a majority of antibiotics,and the ESBLs-or/and AmpC-producing strains had been prevalent widely.Genotypes of ESBLs prevalent in Shanxi province were in accordance with those in other regions of China,whereas the bla_FOX genotype of AmpC detected in our test was relatively less reported in domestic.

This study was aimed to analyze and determine the main active components of total flavonoids in the seeds of Ammopiptanthus mongolicus by high performance liquid chromatography-mass spectrometry (LC-MS) and high performance liquid chromatography (HPLC),and explore the effects of total flavonoids in the seeds of Ammopiptanthus mongolicus on immune function in mice.The main active components in total flavonoids of the seeds of Ammopiptanthus mongolicus was analyzed by LC-MS,and the content of the main active components was determined by standard substance under the optimized conditions of HPLC.The effects of proliferation of splenic lymphocytes and KCs were detected by MTT method,the contents of IL-2,IL-4,NO and NOS were measured by ELISA Kit,the antibody-producing cells and hemolysin in mice serum were explored by quantitative hemolytic spectrophotometry and HC₅₀ determination.The results showed that the main compounds of total flavonoids from the seeds of Ammopiptanthus mongolicus were manshank,7,3'-dihydroxy-4'-methoxyflavone,glycitein and amentoflavone,among them,the content of manshank was 52.48%,and the content of 7,3'-dihydroxy-4'-methoxyflavone was 11.20%.The total flavonoids from the seeds of Ammopiptanthus mongolicus had obvious promoting effect on the proliferation of splenic lymphocytes and the production of cytokines,especially at 20 μg/mL total flavonoids,the proliferation rate of splenic lymphocytes and the secretion of IL-2 reached 217.62% and 159.661 pg/mL,respectively,which were extremely significantly higher than that of blank control group (P<0.01);At 40 μg/mL total flavonoids,the secretion of IL-4 in splenic lymphocytes reached 149.274 pg/mL,which was extremely significantly higher than that of blank control group (P<0.01).The total flavonoids from the seeds of Ammopiptanthus mongolicus also significantly promoted the proliferation and NO and NOS secretion of KCs cells.At 60 μg/mL total flavonoids,compared with blank control group,the proliferation rate of KCs cells was 67.77%,the secretion of NO and NOS reached 180.106 and 29.942 μmol/L (P<0.01),respectively.In addition,the total flavonoids could increase the contents of antibody-producing cells and hemolysin in mice serum.The results suggested that the main compounds of total flavonoids from the seeds of Ammopiptanthus mongolicus were manshank and 7,3'-dihydroxy-4'-methoxyflavone.The total flavonoids from the seeds of Ammopiptanthus mongolicus could obviously promote the proliferation of immunocompetent cells,the production and release of related immune cytokines and antibody in mice.

In order to investigate the effect of Cynanchum auriculatum polysaccharide (CAP) on the resistance to Newcastle disease virus (NDV) in vitro,the polysaccharides from Cynanchum auriculatum was extracted by water and alcohol precipitation,phenol sulfuric acid and infrared spectroscopy were used to detect CAP.The safe concentration of CAP on chicken embryo fibroblast (CEF) was determined by MTT method,the safe concentration of CAP was set up to 625 μg/mL,and 5 concentrations in the safe concentration range (39.06 to 625 μg/mL) were selected.Each CAP and NDV were added with three kinds of dosing methods (add CAP and then inoculate the virus,inoculate the virus and then add CAP,CAP and the virus-sensing) to detect the blocking,inhibition and direct effect of CAP on NDV.The results showed that CAP₅₀,CAP₃₀,CAP_t,CAP₈₀ and CAP₇₀ could significantly inhibit the ability of NDV to infect CEF after the virus was first added group and the CAP and the virus-sensing group.The inhibition rates of CAP₅₀,CAP₃₀,CAP_t,CAP₈₀ and CAP₇₀ were 131.23%,110.12%,108.15%,100.24%,93.07% and 61.76%;43.73%,44.51%,29.02% and 37.45%,respectively.The addition of CAP₅₀ and CAP₃₀ in CAP followed by the virus group could significantly inhibited NDV (P<0.05) with 15.82% and 8.33% inhibition rate under the blocking condition,and the other groups had no inhibitory effect on NDV within the safe concentration range.In conclusion,the CAP had better anti-NDV activity,and the inhibition and direct killing effect on NDV was stronger than the blocking effect.CAP₅₀ and CAP₃₀ had stronger anti-NDV activity and could be used as research materials to study further.

In order to explore the pathogenicity and drug resistance of Bacillus cereus isolated from fish and guide scientific drug use,morphological observation,physiological and biochemical characteristics analysis,16S rDNA gene amplification and phylogenetic tree construction were used to identify the isolated bacteria.Artificial infection test was used to determine the pathogenicity of the isolated bacteria,drug resistance gene detection and drug sensitivity test were used to determine the drug resistance of the strain.The results showed that the isolated strain formed irregular and non-smooth milky-white colonies,which were aerobic Gram-positive bacteria.The biochemical reactions such as glucose,nitrate and gelatin liquefaction were positive,while the biochemical reactions such as xylose,arabinose and mannitol were negative.The length of 16S rDNA gene fragment was 1 457 bp.In phylogenetic tree,the isolated strain had the closest relationship with Bacillus cereus RTR strain,and the homology of the isolated strain and Bacillus cereus reached more than 99%,so the isolated strain was identified as Bacillus cereus.Artificial infection test showed that the isolated strain had certain pathogenicity to Pelteobagrus fulvidraco.The results of resistance gene detection showed that two kinds of resistance genes (Sul1 and Sul2) were detected in the isolated strain.Drug sensitivity test showed that the strain was sensitive to gentamicin,piperacillin and minocycline,moderately sensitive to cefoperazone,and resistant to penicillin,ceftriaxone and ampicillin.The results provided scientific reference for the effective prevention and control of Bacillus cereus infection.

In order to provide a new safe and reliable method for simultaneous detection of florfenicol (FF) and its metabolite florfenicol amine (FFA) residues in animal-derived foods,this study established an accelerated solvent extraction/ultra-high performance liquid chromatography-fluorescence detector (ASE/UPLC-FLD) method for the determination of FF and FFA in pork.Samples were concentrated after accelerated solvent extraction,fat was removed by n-hexane saturated with ethylacetate,then concentrated and re-dissolved before analysis in UPLC-FLD system.The results showed that FF and its metabolite FFA had a good linear relationship in the range of 9.8 to 400 and 3.2 to 400 μg/kg,respectively,and the coefficient of determination (R²) were 0.9993 and 0.9999,respectively.The limit of detection (LOD) and the limit of quantification (LOQ) of FF were 3.3 and 9.8 μg/kg,for FFA were 1.2 and 3.2 μg/kg,respectively.The average recoveries of the samples at LOQ,0.5MRL,1.0MRL and 2.0MRL spiked levels ranged from 82.92% to 96.72%,and the relative standard deviations were below 3.11%.In summary,the ASE extraction method had many advantages such as high extraction efficiency,time saving,environmental protection and so on.The UPLC-FLD method had high efficiency,sensitivity and recovery,and it provided a new method for simultaneous determination of FF and FFA residues in animal-derived food and it was a new method fitting on meets testing standards.

In order to identify the pathogen of a serious disease occurred in Mauremys sinensis,a dominant bacteria was isolated from the diseased Mauremys sinensis.It was identified by bacterial isolation and culture,Gram staining microscopy,biochemical test,16S rRNA sequence analysis,animal regression test and drug susceptibility.The isolate was gray-white,opaque,round,neat edge,and had wet and smooth surface with middle protruding colonies in general nutrient agar medium,and orange-red,round,smooth colonies in SS agar medium.Gram staining microscopy showed that the isolate was Gram-negative,short rod-shaped,obtuse round at both ends of the body,single or double-existent.Biochemical tests showed that the isolate was mobilized with positive reactions such as glucose,sucrose,galactose,maltose,fructose,arginine dihydrolase,ornithine decarboxylase,D-glucose acid production,D-glucose gas production,gelatin hydrolysis,oxidase,indole and V-P test.The sequence analysis of 16S rRNA showed that the isolate was clustered with Aeromonas veronii registered in GenBank,and had 99% homology with that reference strains.The animal regression test showed that HNJ002 was highly pathogenic to Mauremys sinensis.Drug sensitivity test showed that the isolate was sensitive to most antibacterial,moderately sensitive to roxithromycin,tobramycin and neomycin,and only resistant to a few antibiotics such as ampicillin,penicillin and clindamycin.This study would provide a scientific basis for the prevention and treatment of Aeromonas veronii in Mauremys sinensis.

Mycotoxins are metabolites produced by the growth of fungi in food or feed,these low molecular weight compounds are naturally occurring and also unavoidable.Fungi enter the food chain through two ways:On the one hand,they can enter the food chain directly from plant food components contaminated with mycotoxins;On the other hand,they can enter the food chain through indirect contamination of the growth of poisonous fungi in food.Mycotoxins are widely found in mature corn,grains,soybeans,sorghum,peanuts and forage crops.Eating foods or feeds contaminated with mycotoxins can cause acute or chronic toxicity to humans and animals.In addition to the adverse effects of direct consumption of food and feed contaminated by mycotoxins,mycotoxins also have public health problems caused by intake of animal-derived foods,such as meat,milk or eggs,residues or metabolites containing mycotoxins.Although more than 400 mycotoxins have been identified,6 toxins are widely found in food:Aflatoxin,ochratoxin,zearalenone,fumonisin,vomiting toxin and T-2 toxin,which have caused persistent food safety problems around the world.This review summarizes the toxicity of 6 mycotoxins,focusing on the recent advances in the detection of these biomyxins by electrochemical biosensors,aiming to prospectively develop their prospects for mycotoxin detection through a series of summaries.

In order to study the metabolism of artemisinin (ART) in chickens,the LC-MS/MS method was established for the determination of ART in chicken plasm.The electrospray ionization (ESI) source was used as the ion source,and artemether (ARM) was the internal standard(IS).The[M+Na]⁺ quasi ion peaks of ART and ARM were chosed for secondary mass spectrometry.Two pairs of ions,m/z 305.2→151.3 (ART) and m/z 321.0→163.1 (ARM),were selected for quantitative analysis by multi-monitoring reaction (MRM).It was concluded that there was a good linear relationship in the range of 10-1 000 ng/mL (R²=0.9997).In this method,the minimum quantitative limit was 10 ng/mL and the recovery rate was between 90% and 105%.Meanwhile,the intra-day and inter-day precision (RSD) were less than 15%.Thirty-day-old chickens were divided into six groups randomly according to the three doses (15,40 and 100 mg/kg) and the number of oral times (one or eleven).The one-time groups were administered ART at the 11th time,and the solvent was orally administered 10 times before.Eleven-time groups were fed ART for 11 times.After the last oral medication,300 μL blood of each chicken was collected in anticoagulant tube at 10 min,30 min,1 h,1.5 h,2 h,2.5 h,3 h,4 h,6 h and 8 h.The results showed that the concentration of ART in the blood of the chicken which were administered for one time (15,40 and 100 mg/kg) could reach the C_max at 1 or 1.5 h.And the metabolism of ART in chickens could be accelerated by orally administered for more times and has a dose-dependent manner.In conclusion,the method established in this study was simple,sensitive and reproducible for the determination of ART in chicken plasma.There was a self-induced metabolism of ART in chickens,meanwhile,higher the dose,faster the metabolism.

The swine bacterial infectious diseases in China whose outbreak situation has change gradually in recent years is a serious threat to pig-breeding industry and public health security.The rapid and accurate detection of pathogens is a key point to prevent and control swine diseases.Traditional techniques,such as isolation and cultivation of pathogens,conventional PCR and early immunological assays has been constantly changed and evolved to be more accurate and efficient with the development of biotechnology and the intersections of different disciplines.This article highlighted several new detection technologies:Real-time quantitiative PCR,loop-mediated isothermal amplification,sandwich DNA hybridization assay which based on nucleic acid molecular biology;Immune colloidal gold technique,time resolved fluorescence immunoassay,immunomagnetic bead separation which base on immunology;Mass spectrograph detection of bacteria which base on protein molecular biology and The POCT technology appropriated to on-site detection.The advantages and disadvantages of these new technologies wre analyzed,which would be helpful to popularize this detection in different institutions and research departments to select in order to make them play a more important role in the detection and surveillance of swine bacterial infectious diseases.

In order to study the stability of the rifaximin uterine injection,and initially determined its quality guarantee period,three batches of rifaximin were used to test the stability of uterine injectors,including the influencing factors test,the high temperature accelerated test and long term stability test,the content of rifaximin and its related substances were determined by high performance liquid chromatography (HPLC).Thus,the quality guarantee period of rifaximin uterine injection was preliminarily determined.Referring to the relevant technical specifications,rabbit vaginal mucous membrane irritation test was studied.The results showed that the contents of the related substances were not more than 3% according to the area normalization method,the properties,sedimentation and particle size of the injector did not change significantly,and the change of the content of rifaximin in the principal component was 90.0% to 110.0%,the validity of this product was determined to be two years.In rabbit vaginal mucous membrane irritation test,the results showed that erythema and edema were not been found on rifaximin uterus implants administration site skin and saline control site skin,and the score was 0 point.In the vaginal mucous membrane irritation test,leukocyte infiltration,hyperemia and edema were not found in vaginal mucosa of test and control groups,vaginal mucous membrane irritation index was 0.25.So,rifaximin uterus injection had no irritation on rabbit vaginal mucous membrane.This product had good clinical safety and could be further promoted and used.

In order to evaluate the efficacy of a Chinese herbal compound prescription against pathogenic Escherichia coli in chicken,which made of four tradition Chinese medicines including Dichrocephala benthamii,Mesona blumes,Polygonum macrophyllum and Nothopanax dalavayi,firstly,the disease materials were isolated and identified,then,the drug susceptibility test was used to detect the diameter of drug bacteriostasis circle,the safety of traditional Chinese medicine was also detected by mice,finally,artificial infection model of avian pathogenic Escherichia coli and the treatment test were implemented,the traditional Chinese medicine compound preparation was implemented for each chicken by oral administration with high,medium and low doses of 1,0.5 and 0.25 g/mL,respectively.The results showed that red colonies were presented on McConkay agar medium,red and small bacilli were observed by Gram staining and microscopic examination.Biochemical identification showed that glucose,maltose,lactose,sucrose,mannitol,indole and MR were positive,ferric trisaccharide produced acid and gas,citrate,VP and hydrogen sulfide were negative.The isolated bacteria were detected by PCR,the specific bands of 1 466 bp were obtained for both the detected gene and the control gene.The diameters of the bacteriostatic circles of traditional Chinese medicine and ciprofloxacin were 15.2 and 18.3 mm,respectively.All mice were survived,there was no significant difference between the average weight gain of mice in each experimental group and blank control group (P>0.05),no adverse reactions were observed for mice after oral administration with traditional Chinese medicine.The survival rates in high,medium and low dose groups of traditional Chinese medicine,ciprofloxacin,infected untreated and negative control were 96.7%,93.3%,63.3%,93.3%,50.0% and 100%,respectively.The rates of recovery in high,medium and low dose groups of traditional Chinese medicine and ciprofloxacin were 96.7%,93.3%,63.3% and 93.3%,respectively.The mean weight gain and weight of thymus gland in high and medium dose groups of traditional Chinese medicine were no significant differences (P>0.05),but were significantly different from those in low dose group of traditional Chinese medicine (P<0.05).The weight of spleen in high and medium dose groups of traditional Chinese medicine and ciprofloxacin were no significant differences (P>0.05),but which were significantly different from those in low dose group of traditional Chinese medicine and infected control group (P<0.05).The results demonstrated that efficacies of 1 and 0.5 g/mL of traditional Chinese medicine against Escherichia coli were excellent,and similar with that of ciprofloxacin.The therapeutic efficacy of low dose of traditional Chinese medicine was non-obvious.